The Aedes aegypti densonucleosis virus (Aedes DNV or AeDNV) is a member of the heterogenous group of densoviruses (family Parvoviridae) that infect invertebrates (mostly insects) (Siegl et al., 1985, Intervirology, 23:61-73; Tijssen, P., 1990, Handbook of Parvoviruses, P. Tijssen, Ed., CRC Press, Boca Raton, Fla.).
Parvoviridae are small (18-28 nm) icosahedral nonenveloped viruses containing single-stranded linear DNA genomes of 4 to 6 kb in length. A typical parvoviral genome consists of a long coding region flaked by terminal hairpin structures of 115 to 365 nucleotides (nt) at each end. The coding region of a parvovirus genome is occupied almost entirely by two long consecutive open reading frames (ORFs). The left ORF codes for a major nonstructural protein (NS1) containing a sequence with a nucleotide binding motif and the right ORF encodes the capsid proteins (VPs) (Cotmore and Tattersal 1987, Advances in Virus Research 33:91-174). Smaller ORFs encoding additional non-structural proteins are also found in the genome of most parvoviruses. Usually, only one strand ("plus") of the genome of mammalian parvoviruses is used to code for virus proteins (Cotmore and Tattersal 1987). The densovirus infecting Junonia coenia however, has coding sequences for its structural proteins located on the "minus" strand (Giraud et al. 1992, Virology 186:207-218; Dumas et. al., 1992, Virology 191:202-222).
AeDNV was originally isolated from a laboratory colony of A. aegypti (Lebedeva et al., 1973, Acta Virologica 17:253-256). The virus is infectious to mosquito larvae of the genera Aedes, Culex, and Culiseta when introduced into the water in which they are reared. Anopheles larvae can be infected only by injection. The virus can infect all stages of larvae, pupae, and the adults of both sexes. Almost all tissues of larvae show cytopathological evidence of infection including fat body, muscles, hindgut, imaginal disks, malpighian tubules, ovaries, and others (Buchatsky, L. P., 1989, Diseases of Aquatic Organisms 6:145-150). The virus particles are typical of a parvovirus, with a diameter of 18-20 nm. They are resistant to organic solvents such as chloroform, to pH between 4 and 11, and to prolonged incubation at 50.degree. C. (Buchatsky 1989).
The sequence of the cloned AeDNV viral genome shows many characteristics typical of a parvovirus genome (Afanasiev et al. 1991, Virology 185:323-336). The virion DNA is about 4000 nt in length with palindromic sequences capable of forming Y-like secondary structures at each end. The palindromic sequences contain cis-acting signals required for DNA replication and encapsidation (Diffoot et al., 1989, J. Virol. 63:3180-3184). The leftward position of the AeDNV genome is occupied by a large ORF (left ORF) that encodes the AeDNV NS1 protein of about 97.5 kDa. The right ORF of the AeDNV genome could encode a capsid protein (VP) of about 40 kDa, which is in good agreement with the sizes of proteins observed from purified virus (approximately 40 and 38 kDa). Presumably the smaller protein is derived from the larger by proteolytic cleavage of a small peptide fragment from the amino terminus, as occurs in many of the mammalian parvoviruses. Besides having the smallest genome and the shortest VPs among known parvoviruses, AeDNV is also unique in the following features: a second ORF (mid ORF) capable of encoding a protein of 41 kDa is located completely within the left ORF and there is also an ORF in the negative strand capable of encoding a protein of 26 kDa. Little is known, however, about the virus gene expression, including whether all of the ORFs observed in the AeDNV genome are transcribed and translated into proteins (Afanasiev, 1991).
Another mosquito densovirus (AaPV) has been isolated recently from Aedes albopictus C6/36 cell line (Jousset et al. 1993, Virus Research 29:99-114). The analysis of the AaPV genome showed that this virus is closely related to AeDNV (Boublik et al. 1994, Virology 200:752-763). These viruses share 77.3% nucleotide and between 73 and 78% amino acid sequence homologies. The organization of the AaPV genome is also similar to that of AeDNV except that no potential ORF has been found on the minus strand of AaPV (Boublik et al. 1994).
In recent years several types of viruses have been successfully used as vectors for introduction and expression of foreign genes in eukaryotic cells. Parvoviruses exhibit several features that are beneficial for developing such expression vectors (Carter, B. J., 1990, Handbook of Parvoviruses, P. Tijssen, Ed., CRC Press, Boca Raton, Fla., pp. 247-284). The genome of parvoviruses are among the smallest of animal DNA viruses known and hence, easier to handle for cloning and transfection procedures. It has been shown that a cloned parvovirus genome transfected into eukaryotic cells can be rescued from the plasmid vector and replicate as a wild-type virus (Samulski et al. 1982, Proc. Natl. Acad. Sci. USA 79:2077-2081). The promoter for the parvovirus capsid gene is very efficient for expression of foreign genes, especially when trans-activated (Rhode, S. L., 1985, Journal of Virology 55:886-889).
Parvovirus vectors have been used to introduce and express different foreign genes in mammalian cells. (Tratschin et al., 1984, Molecular and Cellular Biology 5:3251-3260; Hermonat and Muzyczka 1984, Proc. Natl. Acad. Sci. USA 81:6466-6470; Carter 1990; Muzyczka, N., 1992, Current Topics in Microbiology and Immunology 158:97-129; Russel et al., 1992, Journal of Virology 66: 2821-2828). Recombinant parvovirus genomes carrying a foreign gene can be encapsidated into infectious particles if complemented in trans with capsid and the nonstructural (NS1 and NS2) proteins. Because the genome of a human parvovirus, adeno-associated virus (AAV), is able to integrate into cell chromosomes, special attention was given to develop a stable expression vector from this virus (Cheung et al., 1980, Journal of Virology 33:739-748; Hermonat and Muzyczka 1984, Proc. Natl. Acad. Sci. USA 51:6466-6470. Tratschin et al. 1985, Molecular and Cellular Biology 5:3251-3260; Lebkowski et al. 1988, Molecular and Cellular Biology 8:3988-3996).
Densoviruses present an attractive opportunity to develop expression vectors for insects. Unfortunately, this group has been studied much less than mammalian parvoviruses. The cloned Junonia coenia densovirus (JcDNV) has been used to express .beta.-gal when fused into the one open reading frame (ORF1) located on the plus DNA strand and transfected into insect SPC-SL 52 cells. However, because there is only one ORF on the plus strand of the JcDNV genome, the number of heterologous genes which can be inserted into a JcDNV expression vector is limited.
In addition, virions produced in the JcDNV expression system are poorly infectious in SPC-SL 52 transfected cells. The poor infectivity prevents cell to cell spreading of the virus and results in a rapid drop of virus titer after few passages. (Giraud et al., 1992).
Galleria mellonella densovirus (GmDNV), closely related to JcDNV has been exploited recently as an expression vector injected into larvae of its natural host, Galleria mellonella (greater wax moth). However, a major impediment to the study of GmDNV gene expression is the absence of a tissue culture host for the virus. Tal and Attathom 1993, Archives of Insect Biochemistry and Physiology 22:345-356).
Baculovirus has also been used as an expression system in insect cells. A drawback of the Baculovirus expression system is that the Baculovirus lyse their insect host cells causing reductions in yield and other complications in isolating and purifying recombinantly expressed protein. Lysis of the Baculovirus host cells also prevents its use in the generation of DNA libraries. A further drawback of the baculovirus system is that direct cloning is not possible in this system.
Thus, there is a need for a densoviral expression system for use in host cells which is highly infectious and non-lytic. Also desired is a densoviral expression system which can express several different heterologous genes in one vector. Integration of the densoviral genome and heterologous coding sequence into the host tell genome is also desired so that stably expressing cell lines can be generated. Finally, host cell/expression vector combinations in which the densovirus virions are highly infectious without being cytopathic are needed.